Sequential binding of αVβ3 and ICAM-1 determines fibrin-mediated melanoma capture and stable adhesion to CD11b/CD18 on neutrophils

Fibrin (Fn) deposition defines several type 1 immune responses, including delayed-type hypersensitivity and autoimmunity in which polymorphonuclear leukocytes (PMNs) are involved. Fn monomer and fibrinogen are multivalent ligands for a variety of cell receptors during cell adhesion. These cell receptors provide critical linkage among thrombosis, inflammation, and cancer metastasis under venous flow conditions. However, the mechanisms of Fn-mediated interactions among immune cells and circulating tumor cells remain elusive. By using a cone-plate viscometer shear assay and dual-color flow cytometry, we demonstrated that soluble fibrinogen and Fn had different abilities to enhance heterotypic aggregation between PMNs and Lu1205 melanoma cells in a shear flow, regulated by thrombin levels. In addition, the involvement of integrin α(v)β(3), ICAM-1, and CD11b/CD18 (Mac-1) in fibrin(ogen)-mediated melanoma-PMN aggregations was explored. Kinetic studies provided evidence that ICAM-1 mediated initial capture of melanoma cells by PMNs, whereas α(v)β(3) played a role in sustained adhesion of the two cell types at a shear rate of 62.5 s(-1). Quantitative analysis of the melanoma-PMN interactions conducted by a parallel-plate flow chamber assay further revealed that at a shear rate of 20 s(-1), α(v)β(3) had enough contact time to form bonds with Mac-1 via Fn, which could not otherwise occur at a shear rate higher than 62.5 s(-1). Our studies have captured a novel finding that leukocytes could be recruited to tumor cells via thrombin-mediated Fn formation within a tumor microenvironment, and α(v)β(3) and ICAM-1 may participate in multistep fibrin(ogen)-mediated melanoma cell adhesion within the circulation.     

Kinetics analysis of binding between melanoma cells and neutrophils

It has been determined previously that polymorphonuclear leukocytes, or PMNs, can facilitate melanoma cell extravasation through the endothelium under shear conditions. The interactions between melanoma cells and PMNs are mediated by the beta2-integrins expressed by PMNs and intercellular adhesion molecules (ICAM-1) expressed on melanoma cells. In this study, the kinetics of these interactions was studied using a parallel plate flow chamber. The dissociation rates were calculated under low force conditions for ICAM-1 interactions with both beta2-integrins, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), together and separately by using functional blocking antibodies on PMNs. The kinetics of PMNs stimulated with IL-8 was also determined. It was concluded that the small number of constitutively expressed active beta2-integrins on PMNs are sufficient to bind to ICAM-1 expressed on melanoma cells and that the intrinsic dissociation rate for these adhesion molecules appear to be more dependent on what method is used to determine them than on what cells express them.     

Shear stress and shear rate differentially affect the multi-step process of leukocyte-facilitated melanoma adhesion

Previous studies have shown that neutrophils (PMNs) facilitate melanoma cell extravasation [M.J. Slattery, C. Dong, Neutrophils influence melanoma adhesion and migration under flow conditions, Intl. J. Cancer 106 (2003) 713–722] Little is known, however, about the specific interactions between PMNs, melanoma and the endothelium (EC) or the molecular mechanism involved under flow conditions. The aim of this study is to investigate a “two-step adhesion” hypothesis that involves initial PMN tethering on the EC and subsequent melanoma cells being captured by tethered PMNs. Different effects of hydrodynamic shear stress and shear rate were analyzed using a parallel-plate flow chamber. Results indicate a novel finding that PMN-facilitated melanoma cell arrest on the EC is modulated by shear rate, which is inversely-proportional to cell–cell contact time, rather than by the shear stress, which is proportional to the force exerted on formed bonds. β₂ integrins/ICAM-1 adhesion mechanisms were examined and the results indicate LFA-1 and Mac-1 cooperate to mediate the PMN–EC–melanoma interactions under shear conditions. In addition, endogenously produced IL-8 contributes to PMN-facilitated melanoma arrest on the EC through the CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) on PMN. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines and PMN–melanoma adhesion in the recruitment of metastatic cancer cells to the EC.     

Melanoma cell extravasation under flow conditions is modulated by leukocytes and endogenously produced interleukin 8

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for the migration of tumor cells out of the vascular system to establish metastases. Innate immune system processes can potentially promote tumor progression through inflammation dependant mechanisms. White blood cells, neutrophils (PMN) in particular, are being studied to better understand how the host immune system affects cancer cell adhesion and subsequent migration and metastasis. Melanoma cell interaction with the EC is distinct from PMN-EC adhesion in the circulation. We found PMN increased melanoma cell extravasation, which involved initial PMN tethering on the EC, subsequent PMN capture of melanoma cells and maintaining close proximity to the EC. LFA-1 (CD11a/CD18 integrin) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, while Mac-1 (CD11b/CD18 integrin) affected prolonged PMN-melanoma aggregation. Blocking E-selectin or ICAM-1 (intercellular adhesion molecule) on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. Results indicated a novel finding that PMN-facilitated melanoma cell arrest on the EC could be modulated by endogenously produced interleukin-8 (IL-8). Functional blocking of the IL-8 receptors (CXCR1 and CXCR2) on PMN, or neutralizing soluble IL-8 in cell suspensions, significantly decreased the level of Mac-1 up-regulation on PMN while communicating with melanoma cells and reduced melanoma extravasation. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines, and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the endothelium in the microcirculation, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.     

Tumor Cell Extravasation Mediated by Leukocyte Adhesion is Shear Rate Dependent on IL-8 Signaling^*

To complete the metastatic journey, cancer cells have to disseminate through the circulation and extravasate to distal organs. However, the extravasation process, by which tumor cells leave a blood vessel and invade the surrounding tissue from the microcirculation, remains poorly understood at the molecular level. In this study, tumor cell adhesion to the endothelium (EC) and subsequent extravasation were investigated under various flow conditions. Results have shown polymorphonuclear neutrophils (PMNs) facilitate melanoma cell adhesion to the EC and subsequent extravasation by a shear-rate dependent mechanism. Melanoma cell-PMN interactions are mediated by the binding between intercellular adhesion molecule-1 (ICAM-1) on melanoma cells andb2integrins on PMNs. In addition, the fluid convection affects the extent of activation ofb2integrins on PMNs by endogenously secreted interleukin 8 (IL-8) within the tumor microenvironment. Results also indicate that shear rate affects the binding kinetics between PMNs and melanoma cells, which may contribute to the shear-rate dependence of melanoma extravasation in a shear flow when mediated by PMNs.     

The integrins Mac-1 and alpha4beta1 perform crucial roles in neutrophil and T cell recruitment to lungs during Streptococcus pneumoniae infection

Neutrophils and T cells play an important role in host protection against pulmonary infection caused by Streptococcus pneumoniae. However, the role of the integrins in recruitment of these cells to infected lungs is not well understood. In this study we used the twin approaches of mAb blockade and gene-deficient mice to investigate the relative impact of specific integrins on cellular recruitment and bacterial loads following pneumococcal infection. We find that both Mac-1 (CD11b/CD18) and α(4)β(1) (CD49d/CD29) integrins, but surprisingly not LFA-1 (CD11a/CD18), contribute to two aspects of the response. In terms of recruitment from the circulation into lungs, neutrophils depend on Mac-1 and α(4)β(1), whereas the T cells are entirely dependent on α(4)β(1). Second, immunohistochemistry results indicate that adhesion also plays a role within infected lung tissue itself. There is widespread expression of ICAM-1 within lung tissue. Use of ICAM-1(-/-) mice revealed that neutrophils make use of this Mac-1 ligand, not for lung entry or for migration within lung tissue, but for combating the pneumococcal infection. In contrast to ICAM-1, there is restricted and constitutive expression of the α(4)β(1) ligand, VCAM-1, on the bronchioles, allowing direct access of the leukocytes to the airways via this integrin at an early stage of pneumococcal infection. Therefore, integrins Mac-1 and α(4)β(1) have a pivotal role in prevention of pneumococcal outgrowth during disease both in regulating neutrophil and T cell recruitment into infected lungs and by influencing their behavior within the lung tissue itself.     

Two-dimensional kinetics of beta 2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta 2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta 2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta 2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.     

Distinct role of hydrodynamic shear in leukocyte-facilitated tumor cell extravasation

Previously, we found polymorphonuclear neutrophils (PMNs) increased melanoma cell extravasation under flow conditions (Intl J Cancer 106: 713–722, 2003). In this study, we characterized the effect of hydrodynamic shear on PMN-facilitated melanoma extravasation using a novel flow-migration assay. The effect of shear stress and shear rate on PMN-facilitated melanoma extravasation was studied by increasing the medium viscosity with dextran to increase shear stress independently of shear rate. Under fixed shear rate conditions, melanoma cell extravasation did not change significantly. In contrast, the extravasation level increased at a fixed shear stress but with a decreasing shear rate. PMN-melanoma aggregation and adhesion to the endothelium via ₂-integrin/intracellular adhesion molecule-1 (ICAM-1) interactions were also studied. Lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, whereas Mac-1 (CD11b/CD18) affected prolonged PMN-melanoma aggregation. Blockage of E-selectin or ICAM-1 on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. We have found PMN-melanoma adhesion is correlated with the inverse of shear rate, whereas the PMN-endothelial adhesion correlated with shear stress. Interleukin-8 (IL-8) also influenced PMN-melanoma cell adhesion. Functional blocking of the PMN IL-8 receptors, CXCR1 and CXCR2, decreased the level of Mac-1 upregulation on PMNs while in contact with melanoma cells and reduced melanoma extravasation. We have found PMN-facilitated melanoma adhesion to be a complex multistep process that is regulated by both microfluid mechanics and biology. neutrophil; melanoma; shear stress; shear rate; ₂-integrins; intracellular adhesion molcule-1; CXCR1/2; adhesion; migration     

Integrin VLA-4 enhances sialyl-Lewisx/a-negative melanoma adhesion to and extravasation through the endothelium under low flow conditions

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewis(x/a)-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of alpha(4)beta(1) (VLA-4) on sialyl-Lewis(x/a)-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, beta(2)-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and beta(2)-integrins on PMNs were necessary for facilitating the melanoma extravasation process.     

Determining beta2-integrin and intercellular adhesion molecule 1 binding kinetics in tumor cell adhesion to leukocytes and endothelial cells by a gas-driven micropipette assay

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding β(2)-integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attributed to the high expression of ICAM-1 on WM9 cells. These results proposed an alternative mechanism, where WM9 melanoma cells adhere first with PMNs near vessel-wall regions and then bind to endothelial cells via PMNs under blood flow. In contrast, the adhesions between human MDA-MB-231 metastatic breast carcinoma cells and PMNs showed a comparable cellular binding affinity to PMN-HPMEC pair because the ICAM-1 expressions on MDA-MB-231 cells and HPMECs are similar. Furthermore, differences were observed in the intrinsic forward and reverse rates of the β(2)-integrin-ICAM-1 bond between PMN-TC and PMN-EC pairs. This GDMAT assay enables us to quantify the binding kinetics of cell adhesion molecules physiologically expressed on nucleated cells. The findings also further the understanding of leukocyte-facilitated tumor cell adhesion from the viewpoint of molecular binding kinetics.     

Study on intracellular trafficking of Mac-1 by direct visualization

Mac-1 (macrophage differentiation antigen asso-ciated with complement three receptor function), alsonamed CD11b/CD18, an adhesion molecule belongingresting leukocytes and at the surface of activated leu-kocytes (macrophages and neutrophils). Mac-1 playsan important role in the migration, chemotaxis and 2 Methodsphagocytosis of leukocytes[1]. Although there have 2.1 Observation of PMA-stimulated Mac-1 traffick-been several reports on the role of Mac-1 in neutrophil ingadh…     

Study on intracellular trafficking of Mac-1 by direct visualization

Previously, we constructed DNA vectors containing cDNAof Mac-1 subunits (CD11b or CD18b) fused with fluorescence protein (FP). cDNA fragments and the DNA constructs were then transfected into CHO cells (as CHO-Mac-1-FP). The structure and function of Mac-1-FP obtained from the CHO-Mac-1-FP cells are nearly identical to that expressed in wild type leukocytes. In the present study, the intracellular trafficking of Mac-1 was visualized directly by monitoring the fluorescent intensities of YFP-CD18 and PE-conjugated monoclonal antibody against CD11b under a confocal microscope in CHO-Mac-1-FP cells. The results indicate that: (i) although Mac-1 was not detected in the cell membrane at resting state, it had been translocated and clustered into the cell membrane by 1 h and internalized 2 h after PMA stimulation, at which point the fluorescence intensity began to diminish gradually, probably due to partial degradation of Mac-1. The fluorescence of CD18 and CD11b reappeared on the cell membrane 1 h after re-treatment with PMA, suggesting the recycling of non-degraded Mac-1. (ii) The adhesion rate of CHO-Mac-1-FP to magnetic beads coupled ICAM-1 increased within 4 h after their initial interaction, accompanied by the clustering of Mac-1-FP. After 8 h, the adhesion rate declined and fluorescence also decreased simultaneously. The pattern of change in fluorescence in CHO-Mac-1-FP cells elicited by ICAM-1 beads was similar to that elicited by PMA, suggesting that endocytosis and degradation of Mac-1 occurred after the interaction with ICAM-1. Thus, we conclude that the intracellular trafficking of Mac-1 after activation is associated with membrane translocation, endocytosis, degradation and recycling. These changes are in parallel with the adhesion of CHO-Mac-1-FP cells with ICAM-1, and may be involved in the adhesion and detachment of leukocytes. The detachment of leukocytes may be caused by endocytosis of Mac-1.     

CCN6 enhances ICAM-1 expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN6 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, and Nov) family of matricellular proteins. However, the effects of CCN6 on human chondrosarcoma cells are largely unknown. In this study, we found that CCN6 increased the migration and the expression of intercellular adhesion molecule-1 (ICAM-1) in human chondrosarcoma cells. αvβ3 and αvβ5 integrin monoclonal antibody and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the CCN6-induced increase of the migration and ICAM-1 up-regulation of chondrosarcoma cells. CCN6 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK, and extracellular signal-regulated kinase (ERK). In addition, activator protein-1 (AP-1) inhibitors suppressed the cell migration and ICAM-1 expression enhanced by CCN6. Moreover, CCN6 increased AP-1 luciferase activity and binding of c-Jun to the AP-1 element on the ICAM-1 promoter. Taken together, our results indicate that CCN6 enhances the migration of chondrosarcoma cells by increasing ICAM-1 expression through the αvβ3 and αvβ5 integrin receptor, FAK, MEK, ERK, c-Jun, and AP-1 signal transduction pathway.     

脉络膜黑色素瘤中整合素αvβ3表达的研究

目的：整合素αvβ3整合素家族成员之一,是一类跨膜粘附分子,其在多种肿瘤细胞及新生内皮血管细胞中高表达而在成熟血管内皮、上皮细胞及正常细胞中表达较低或无表达。基于这些特征,整合素αvβ3年来成为分子影像研究的热点之一。鉴于整合素αvβ3人眼脉络膜黑色素瘤中是否存在表达尚不可知,本课题拟研究整合素αvβ3人脉络膜黑色素瘤中表达情况,为以整合素αvβ3基础的分子显像提供理论基础。方法：培养人源脉络膜黑色素瘤细胞株OCM-1,使用蛋白免疫印迹方法检测整合素αvβ3OCM-1中表达水平,并使用免疫组化方法检测人脉络膜黑色素瘤病理切片中整合素αvβ3达分布。结果：蛋白免疫印迹实验表明在OCM-1细胞株中存在整合素αvβ3达,其表达水平介于已知高表达整合素αvβ3头颈鳞癌细胞株HEP-2和较低表达整合素αvβ3头颈鳞癌细胞株CNE-1之间,免疫组化方法检测人脉络膜黑色素瘤病理切片中也存在整合素αvβ3达。结论：人脉络膜黑色素瘤中具有整合素αvβ3达,可以为后期研究基于整合素αvβ3分子显像技术提供依据。     

Sialic acid associated with αvβ3 integrin mediates HIV-1 Tat protein interaction and endothelial cell proangiogenic activation

Sialic acid (NeuAc) is a major anion on endothelial cells (ECs) that regulates different biological processes including angiogenesis. NeuAc is present in the oligosaccharidic portion of integrins, receptors that interact with extracellular matrix components and growth factors regulating cell adhesion, migration, and proliferation. Tat is a cationic polypeptide that, once released by HIV-1(+) cells, accumulates in the extracellular matrix, promoting EC adhesion and proangiogenic activation by engaging α(v)β(3). By using two complementary approaches (NeuAc removal by neuraminidase or its masking by NeuAc-binding lectin from Maackia amurensis, MAA), we investigated the presence of NeuAc on endothelial α(v)β(3) and its role in Tat interaction, EC adhesion, and proangiogenic activation. α(v)β(3) immunoprecipitation with biotinylated MAA or Western blot analysis of neuraminidase-treated ECs demonstrated that NeuAc is associated with both the α(v) and the β(3) subunits. Surface plasmon resonance analysis demonstrated that the masking of α(v)β(3)-associated NeuAc by MAA prevents Tat/α(v)β(3) interaction. MAA and neuraminidase prevent α(v)β(3)-dependent EC adhesion to Tat, the consequent FAK and ERK1/2 phosphorylation, and EC proliferation, migration, and regeneration in a wound-healing assay. Finally, MAA inhibits Tat-induced neovascularization in the ex vivo human artery ring sprouting assay. The inhibitions are specific because the NeuAc-unrelated lectin from Ulex europaeus is ineffective on Tat. Also, MAA and neuraminidase affect only weakly integrin-dependent EC adhesion and proangiogenic activation by fibronectin. In conclusion, NeuAc is associated with endothelial α(v)β(3) and mediates Tat-dependent EC adhesion and proangiogenic activation. These data point to the possibility to target integrin glycosylation for the treatment of angiogenesis/AIDS-associated pathologies.     

Time-dependent loss of Mac-1 from infiltrating neutrophils in the reperfused myocardium

Numerous studies have shown that polymorphonuclear neutrophils (PMNs) infiltrate the myocardium immediately after reperfusion of infarcted tissue. Studies with mAbs in vivo and cellular studies in vitro suggest that PMN-induced injury of the cardiac myocyte involve Mac-1 adhesion to myocyte ICAM-1. In this study we demonstrate that PMNs that have infiltrated the ischemic area begin to lose Mac-1 within the first 3 h. By the fifth hour of reperfusion, minimal CD11b staining is seen on PMNs using immunostaining, whereas CD11a remained unchanged. Immunoreactivity of postreperfusion cardiac lymph with R15.7 (anti-CD18) or MY904 (anti-CD11b) was positive in all animals but not for CD11a (R7.1), indicating a specific loss of Mac-1. Immunoprecipitation with either R15.7 or MY904 resulted in identical peptides (a doublet at 190 kDa and a band at 80 kDa), suggesting that both alpha and beta subunits of Mac-1 heterodimer were released. Immunoprecipitation of control PMN lysates revealed bands of 198 kDa and 91 kDa slightly greater than those from the released Mac-1. An in vitro model of homotypic aggregation showed a similar loss of Mac-1 from PMNs; immunoprecipitates of the supernatant demonstrated peptide bands identical with those found in postischemic cardiac lymph. The appearance of soluble Mac-1 in vitro was prevented by anti-CD18 mAb, R15.7, and also by protease inhibition by PMSF. Thus, in vivo and in vitro, activated PMNs lose Mac-1 in a process that may be dependent upon adhesion and subsequent proteolysis.     

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18)

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac- 1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity- purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1- dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.     

Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.     

A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen

We report that a subpopulation (10%) of the Mac-1 (CD1 1b/CD18) molecules on activated neutrophils mediates adhesion to ICAM-1 and fibrinogen. We describe a novel mAb (CBRM1/5) that binds to an activation-specific neoepitope on a subset of Mac-1 molecules on neutrophils and monocytes after stimulation with chemoattractants or phorobol esters but does not recognize Mac-1 on resting myeloid cells. CBRM1/5 immunoprecipitates a subpopulation of Mac-1 molecules from detergent lysates of neutrophils, binds to immunoaffinity-purified Mac- 1, and localizes to the I domain on the alpha chain of Mac-1. Because CBRM1/5 recognizes a fraction of Mac-1 on activated neutrophils, but still blocks Mac-1-dependent adhesion to fibrinogen and ICAM-1, we suggest that only a small subset of Mac-1 molecules is competent to mediate adhesion.